Juvenile myelomonocytic leukemia (JMML) is a clonal, mixed myeloproliferative/myelodysplastic disorder afflicting young children. Children with neurofibromatosis type 1 have a 500 fold increased incidence of developing JMML. Since 1986 a team of investigators at the University of Alabama at Birmingham (UAB) have been conducting translational research studies in JMML. The UAB team consists of a blend of investigators in basic science and clinical investigations and this mix has allowed this team to play significant leading roles spanning the breadth of JMML research, from basic science pathogenesis investigations, to translational investigations, to clinical protocols and registries. The pathogenesis of JMML has been linked to dysregulated GM-CSF growth factor signal transduction through the Ras signaling pathway, resulting in GM-CSF hypersensitivity, a hallmark of the disease. Potential causative mutations or other genetic abnormalities in three genes, RAS, NF1, and PTPN11, all of which are positioned in the GM-CSF signal transduction pathway appear to account for up to 75% of cases of JMML. These gene abnormalities, approximately 20% for RAS, approximately 25% for NF1, and approximately 30% for PTPN11, appear to be mutually exclusive suggesting that any one abnormality is sufficient for causation. The approximately 25% estimation of NF1 gene abnormalities is based on the utilization of loss of heterozygosity assays and in vitro transcription/translation assays from primary patient samples and EBV-transformed cell lines derived from these samples. Dr. Messiaen is a new recruit to UAB from Belgium where she developed more comprehensive methodologies for NF1 mutation analysis beyond the above-described assays. Further, she has developed new techniques, which obviate the need for EBVcell line generation. The major hypothesis of this proposal is that the estimated 25% incidence of NF1 mutations in JMML may be inaccurately low, and that additional NF1 mutations can be found in the group of JMML patients previously categorized as no known genetic mutation. Patient material to test this hypothesis, and to develop more efficient methodologies which obviate the need for EBV lines, will be obtained from the currently active Children's Oncology Group protocol for JMML. This is a multi-institutional Cooperative Group trial sponsored by the NCI, which is seeking to determine if genetic differences in JMML predict for different prognoses, and/or varying responses to the targeted therapeutics being tested.